The Effect And Molecular Mechanism Of SIRT1 On ChREBP In Diabetic Renal Tubular Epithelial Cells

Objects: Diabetic nephropathy(DN)is the major cause of chronic kidney disease(CKD),leading to end-stage renal disease.Altered lipid metabolism in diabetes mellitus may cause the change of renal structure and function,which is closely related to the occurrence of diabetic nephropathy.The disorder of lipid metabolism is associated with the abnormal lipid metabolism genes.In this study,we use db/db mice and human renal tubular epithelial cells,through regulating the activity and expression of SIRT1,to explore the effect and molecular mechanism of SIRT1 on Ch REBP in diabetic renal tubular epithelial cells.The aim is to provide new ideas for the treatment of diabetic nephropathy.Methods: 1.Effect of SIRT1 on the renal expression of Ch REBP in db/db miceMale db/db mice(10 weeks old)were randomly divided into diabetes group(db/db),SRT1720 treatment group(db/db+SRT)and db/m mice as the same nest control.SRT1720 of 50mg/kg by gavage was administered to db/db mice daily.Operation was performed after 10 weeks,western blot was conducted to detect the protein expression of SIRT1,Ch REBP,p-AMPK,AMPK and PP2 A.Immunohistochemistry was used to detect the expression of Ch REBP.2.Effect and molecular mechanism of SIRT1 on Ch REBP in HG-induced HK-2 cellsCells were cultured in DMEM/F12 medium at 5%CO2 and 37°C.⑴ Cells were divided into normal glucose group(5.6m M glucose,NG),high glucose group(30m M glucose,HG),HG+SRT1720 group(30m M glucose+2.5μM SRT1720,HG+SRT),HG+EX527 group(30m M glucose+10μM EX527,HG+EX527),HG+SRT1720+Compound C group(30m M glucose+2.5μM SRT1720+2μM Compound C,HG+SRT+CC)and incubated for 48 h.Western blot was used to detect the protein expressions of SIRT1,Ch REBP,p-AMPK,AMPK,LKB1,PP2 A,CPT-1,PPARα,LDLR and FAS in HK-2 cells.After the nuclear and cytoplasmic proteins were isolated,Western blot was conducted to examine the protein expression of Ch REBP in nucleus and in cytoplasma,respectively.The protein expressions of SIRT1 and Ch REBP were assessed by immunofluorescence in HK-2 cells.Oil red O staining was used for the cellular lipid droplets detection.⑵ Cells were divided into normal glucose group(5.6m M glucose,NG),high glucose group(30m M glucose,HG),HG+AICAR group(30 m M+500μM AICAR,HG+AICAR),HG+Compound C group(30 m M+2 μM Compound C,HG+CC)and incubated for 48 h.Western blot was used to detect the protein expression of p-AMPK,AMPK,Ch REBP,PP2 A,CPT-1,PPARα,LDLR and FAS.After the nuclear and cytoplasmic proteins were isolated,Western blot was conducted to examine the protein expression of Ch REBP in nucleus and in cytoplasma,respectively.Immunofluorescence was used to detect the protein expression of Ch REBP in HK-2 cells.The cellular lipid droplets were observed by Oil red O staining.Results:1.Effect of SIRT1 on the renal expression of Ch REBP in db/db miceIn db/db group,the protein expression of Ch REBP was increased,while the protein expression of SIRT1 and phosphorylation of AMPK was decreased compared with db/m group.SRT1720 enhanced SIRT1 protein expression and phosphorylation of AMPK,however,reduced Ch REBP expression(P<0.05).There was no significant change in the protein expression of PP2 A in each group.Immunohistochemistry showed that the expression of Ch REBP in renal cortex of db/db group was increased and that was significantly decreased in SRT1720 group compared with db/m group(P<0.05).2.Effect and molecular mechanism of SIRT1 on Ch REBP in HG-induced HK-2 cells⑴Effect of SIRT1 on the expression of Ch REBP and proteins involved in lipid metabolism in HG-induced HK-2 cells(1)Compared with NG group,the expression of Ch REBP,LDLR and FAS was increased,while the phosphorylation of AMPK was decreased and the expression of SIRT1,LKB1,CPT-1 and PPARα was decreased in HG group.SRT1720 upregulated the phosphorylation of AMPK and the expression of SIRT1,LKB1,CPT-1 and PPARα,while downregulated the expression of Ch REBP,LDLR and FAS(P<0.05).CC treatment could reverse these changes brought by SRT1720(P<0.05).Compared with HG group,phosphorylation of AMPK and the protein expression of LKB1,CPT-1 and PPARα was decreased in HG +EX527 group(P<0.05),while the protein expression of Ch REBP,LDLR and FAS failed to show further changes.(2)The protein expression of Ch REBP in nucleus was increased and that in cytoplasma was decreased in HG group,compared with NG group.Compared with HG group,the protein expression of Ch REBP in nucleus was decreased and that protein in cytoplasma was increased in SRT group(P<0.05).EX527 treatment decreased the protein expression of Ch REBP in cytoplasma,with no significant difference of Ch REBP expression in nucleus,compared with HG group.(3)Immunofluorescence showed a similar trend with Western blot.(4)Oil red O staining showed that SRT1720 reduced the increased lipid drop induced by high glucose,while EX527 had no significant effect.⑵Effect of AMPK on the expression of Ch REBP and proteins involved in lipid metabolism in HG-induced HK-2 cells(1)Compared with NG group,the expression of Ch REBP,LDLR and FAS was increased,while the phosphorylation of AMPK was decreased and the expression of CPT-1 and PPARα was decreased in HG group.AICAR could increase the phosphorylation level of AMPK and the expression of CPT-1 and PPARα,however decrease the expression of Ch REBP,LDLR and FAS.CC treatment decreased the phosphorylation of AMPK and the expression of CPT-1 and PPARα,increased the expression of Ch REBP but with no significant changes of LDLR and FAS(P<0.05).(2)The protein expression of Ch REBP in nucleus was increased and that in cytoplasma was decreased in HG group,compared with NG group.AICAR treatment suppressed the protein expression of Ch REBP in nucleus and elevated that expression in cytoplasma.The reduction of Ch REBP in cytoplasma and the elevation in nucleus were found after CC treatment(P<0.05).(3)Immunofluorescence exerted a similar trend with Western blot.(4)Oil red O staining showed that AICAR reduced the increased lipid droplets in HK-2 cells.While the lipid droplets was increased in CC group.Conclusions:1.Activation of SIRT1 exerted an inhibition of Ch REBP in the kidney of db/db mice.2.SIRT1-mediated the inhibition of Ch REBP and improvement of lipid deposition was partially via AMPK in HG-induced HK-2 cells.